Traditionally, chimeric constructs that comprise multiple unrelated or heterologous nucleic acid sequences have been constructed by inserting individual insert nucleic acid sequences into a recipient vector that comprises one or more other nucleic acid sequences required for the chimeric construct, an origin of replication and a selectable marker gene that is used to confer a trait for which one can ‘select’ based on resistance to a selective agent (e.g., an herbicide, antibiotic, radiation, heat, or other treatment damaging to cells without the marker gene). Typically, the insertion of an insert nucleic acid sequence into the recipient vector comprises digesting a donor vector in which the insert nucleic acid sequence is contained with one or more restriction enzymes to produce a vector backbone and a fragment with blunt or cohesive ends and comprising the insert nucleic acid sequence. This donor vector will generally comprise its own origin of replication and a selective marker gene, which may be the same as, or different than, the selectable marker gene of the recipient vector. The recipient vector is also digested with one or more restriction enzymes to produce a linearized vector with ends compatible with or matching those of the fragment. The digested donor and recipient vectors are then joined by DNA ligation or topoisomerase joining reactions and recombinant vectors containing the insert nucleic acid sequence, the other nucleic acid sequence(s) and the selectable marker gene are identified by introducing the products of the joining process into host cells (e.g., bacteria) and selecting for those that are resistant to the selective agent through the presence of the selectable marker gene corresponding to the recipient vector.
This conventional strategy has several disadvantages including (1) inefficient restriction enzyme cleavage of the vectors, (2) ligation of the fragment to the backbone of the donor vector, (3) ligation-mediated recircularization of the linearized recipient vector and/or (4) generation of linear concatemers containing multiple vectors and/or multiple inserts, which leads to a significant background of non-recombinant host cells (typically 99%) that do not contain the desired recombinant vector. As such, extensive screening of host cells is required in order to identify those with the desired recombinant vector. While this efficiency may be sufficient for simple subcloning experiments, it is unacceptable for the assembly of chimeric constructs requiring multiple heterologous nucleic acid sequences, which need to be sequentially cloned into recipient vectors to produce those constructs. Accordingly, the above conventional strategy generally requires substantial effort and time for producing a desired chimeric construct.
One traditional approach for reducing the background of non-recombinant host cells is to purify the fragment and/or the linearized vector before ligation, which requires larger amounts of vector than would otherwise be required. However, this approach requires further time-consuming steps and the efficiency of ligation of the purified product(s) is generally reduced by trace amounts of agents used for the purification.
Accordingly, there is a need for a cloning system with improved efficiency in producing recombinant vectors that comprise a plurality of unrelated nucleic acid sequences.